Physiologically balanced, ionized, acidic solution and methodology for use in wound healing

ABSTRACT

Described herein is a physiologically-balanced, acidic solution. Typically the solution is prepared by a chemical reactions or by the electrolysis of a solution comprising a mixture of an inorganic salt to form a physiologically balanced solution. This invention also relates to methods for use of the solutions, including a specialized bandage which may be used in combination with the solutions, or optionally with other topically applied materials. A mixture of inorganic salts and, optionally minerals, is used in order to mimic the electrolyte concentration and mixture of body fluid in an isotonic state. The solution typically comprises of one halide salt of lithium, sodium, potassium, calcium, and other cations. Typically the halide is fluoride, chloride, bromide, or iodide, and most typically chloride. A typical electrolyzed solution of the present invention has a pH within the range of about 2 to about 5, an oxidation reduction potential within the range of about +600 mV to about +1200 mV, and hypohalous acid concentration in the range of about 10 ppm to about 200 ppm. The solution has bactericidal, fungicidal, and sporicidal properties. The composition of the invention is nontoxic and has antibacterial properties, and is useful in any application in which antimicrobial properties are desirable.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/655,493, filed Sep. 3, 2003, which is a continuation-in-part of U.S.application Ser. No. 10/209,681, filed Jul. 30, 2002, which is acontinuation-in-part of U.S. application Ser. No. 10/000,919, filed Nov.2, 2001, which is a divisional of U.S. patent application Ser. No.09/482,159, filed Jan. 12, 2000, all of which are incorporated herein byreference in their entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a physiologically balanced, ionized, acidicsolution that is useful in wound healing and other applications in whichantimicrobial properties are desirable. The ionized solution may beprepared by electrolysis, i.e., it is an electrolyzed solution or byother methods including chemical or physical methods. The solution mayalso be prepared in situ. In addition, the invention relates to amethodology of using the solution of the invention, in a variety ofapplications, for example, a specialized bandage which may be used incombination with the solution or with other solutions or topicallyapplied materials.

2. Brief Description of the Background Art

Various electrolyzed acidic salt solutions, their properties, and theiruses have been described in the art. Several examples are providedbelow.

U.S. Pat. No. 5,622,848, issued Apr. 22, 1997, to Morrow, discloses amicrobicidal solution for in vivo and in vitro treatment of microbialinfections. The solution comprises an electrolyzed saline containingregulated amounts of ozone and active chlorine species, wherein theozone content is between about 5 and 100 mg/L, the active chlorinespecies content is between about 5 and 300 ppm and a pH range from7.2-7.6. The active chlorine species comprises free chlorine,hypochlorous acid, and the hypochlorite ion, as measured by a chlorineselective electrode. The solution is prepared by subjecting a 1% or lesssaline solution to electrolysis under conditions sufficient to producethe desired active ingredients. The solution is preferably utilized atan isotonic saline concentration, and may be adjusted with hypertonicsaline. The solution may be used for in vitro treatment of infectedwhole blood, blood cells, or plasma to reduce contamination, and may beused in the treatment of fluids infected with HIV, hepatitis, and otherviral, bacterial, and fungal agents. The solution may also beadministered to warm-blooded animals, including humans, by intravenousinjection or other modes, for similar purposes.

PCT publication No. WO9934652, published Jul. 8, 1999, of Marais,discloses the use of an electrochemically activated sodiumhypochlorite-free irrigating medium to reduce the proliferation ofbacteria and other microorganisms during tooth root canal. Anion- andcation-containing solutions are obtained by electrolysis of a 10%aqueous NaCl solution. The anion-containing solution is used at a pH ofabout 2-7 and an oxidation reduction potential (ORP) of about +1170 mV;the cation-containing solution is used at a pH of about 7-13 and an ORPof about −980 mV.

X. W. Li et al. (Chinese J. Epidem., 17(2), pp. 95-98, 1996) reported apreliminary study of the microbicidal effect of electrolyzed oxidizingwater. Electrolyzed oxidizing water was shown to completely killStaphylococcus aureus and Escherichia coli within 15 seconds, while 10minutes were required to completely kill all spores of Bacillus subtilusvar. niger. Thirty seconds were needed to destroy the antigenicity ofHBsAg. The oxidation reduction potential and pH values of electrolyzedoxidizing water were not significantly changed when stored for threeweeks at room temperature under air-tight, light-free conditions.

A. Iwasawa et al. (J. Jap. Assoc. Infec. Diseases, 70(9), pp. 915-922,1996) evaluated the bactericidal effect of acidic electrolyzed water onS. aureus, S. epidermidis, and Pseudomonas aeruginosa. At pH 5.0 toapproximately 6.0, three bacterial strains were killed soon after beingexposed to the acidic water containing 50 mg/L chloride, and thechloride concentration reportedly did not change after standing open for6 hours. At pH 2.67 to approximately 2.80, the bactericidal effects wereobserved at a chloride concentration of 5 mg/L, and 80% of the chloridereportedly remained after standing open for 6 hours.

H. Tanaka et al. (J. Hosp. Infect., 34(1), pp. 43-49, 1996) reported onthe antimicrobial activity of superoxidized water. Superoxidized wateris described as “a strong acidic and colorless solution with a highoxidation-reduction potential. The solution having an active chlorineconcentration of 30 ppm, is prepared by mixing a small amount of saltwith tap water in an electrolyser”. The antimicrobial activity ofsuperoxidized water was tested against methicillin-sensitive S. aureus,Serratia marcescens, E. coli, P. aeruginosa, and Burkholderia cepacia.The number of bacteria was reduced below the detection limit followingincubation in superoxidized water for 10 seconds. The bactericidalactivity of superoxidized water was similar to that of 80% ethanol, butsuperior to that of 0.1% chlorhexidine and 0.02% povidone iodine.

Y. Inoue et al. (Artificial Organs, 21(1), pp. 28-31, 1997) reported onthe use of electrolyzed strong acid aqueous solution lavage in thetreatment of peritonitis and intraperitoneal abscess. Peritoneal andabscess lavages were performed using an electrolyzed strong acid aqueoussolution to treat seven patients with peritonitis and intraperitonealabscesses. The period of irrigation in the seven patients ranged from 9to 12 days, with conversion to microorganism negative state observedwithin 3 to 7 days. The authors describe the solution as being “acidicwater that contains active oxygen and active chlorine and possesses aredox potential” and having an active chlorine concentration less than50 ppm.

S. Sekiya et al. (Artificial Organs. 21(1), pp. 32-38, 1997) reported onthe use of electrolyzed strong acid solutions in the treatment ofinfectious skin defects and ulcers using. The clinically applied therapyof electrolyzed strong acid aqueous solutions were found to be effectivein the treatment of infectious ulcers. Sekiya et al. describe the strongaqueous solution (ESAAS) as being “generated by electrolyzing water anda small quantity of salt with a cation transfer filter.”

H. Hayashi et al. (Artificial Organs, 21(1), pp. 39-42, 1997) reportedon the use of electrolyzed strong acid aqueous solutions (ESAAS) in thetreatment of mediastinitis following cardiovascular surgery. Hayashi etal. described ESAAS as being “produced by electrolyzing sodium chloridesolution. ( . . . ) ESAAS is produced by electrolyzing the sodiumchloride solution using an ion-exchange membrane that separates thepositive and negative electrodes. A small amount of sodium chloride isadded to the water to facilitate electrolysis and increase theconcentration of dissolved chloride.” The solution is disclosed ashaving a pH less than 2.7, Cl₂ more than 30 ppm, ORP more than 1100, anddissolved O₂ of more than 20 ppm. The mediastinal wound was left openand irrigated with ESAAS one to three times daily until the infectionwas eradicated. Satisfactory growth of granulation tissue was observedin all patients treated, with no evidence of adverse effectsattributable to ESAAS.

N. Tanaka et al. (Artificial Organs, 23(4), pp. 303-309, April 1999)reported on the use of electrolyzed strong acid aqueous solutions toclean and disinfect hemodialysis equipment. The solutions were found todirectly inactivate bacterial endotoxins, and proved to be moreeconomical than the conventional disinfecting method. The “electrolyzedstrong acid aqueous solutions are disclosed to be “strongly acidic waterwhich is made by electrolyzing tap water containing 500-1000 ppm salt(NaCl>99% pure) in a cell partitioned by a polyester diaphragm. It hasan acidity of 2.3-2.7 pH, more than 1,000 mV in oxidation-reductionpotential and 10-50 ppm in available chlorine.”

J. B. Selkon et al. (J. Hosp. Infec., 41 (1), pp. 59-70, January 1999)evaluated the antimicrobial activity of a new superoxidized water,STERILOX® (Sterilox Medical Limited, 85 E Milton Park, Abingdon, OxonOX14 4RY, UK) for the disinfection of endoscopes. This superoxidizedwater is prepared from a 35.7% NaCl in a 1 to 20 dilution, and isdescribed as being “generated at the point of use by passing a salinesolution over coated titanium electrodes at 9 amps. The productgenerated has a pH of 5.0-6.5 and an oxidation reduction potentialof >950 mV.” The antimicrobial activity of STERILOX® was tested againstMycobacterium tuberculosis, M. avium-intracellulare, M. chelonae, E.coli (including type 0157), Enterococcus faecalis, P. aeruginosa, B.subtilus var. niger spores, methicillin-resistant S. aureus, Candidaalbicans, poliovirus type 2, and human immunodeficiency virus HIV-1.Under clean conditions, freshly generated STERILOX® was found to behighly active against all these microorganisms, giving a 5 log₁₀(99.999%) or greater reduction in 2 minutes or less.

U.S. Pat. No. 6,296,744 assigned to Sterilox Technologies InternationalLimited, discloses an apparatus for the electrochemical treatment of aliquid medium, which allows for the production of a sterilizing solutionas well as the decontamination and purification of liquid mediums fromtoxic organic substances and other impurities. The process utilizessolution having an average salinity of 0.1 to 1.0 g/l and a chlorideconcentration of up to 50 mg/l, and the process is carried out using acurrent of 500 to 1000 mA with potential difference of 10-12 volts. Thepatent also discloses that the optimum pH parameters foranodically-treated water are 6-7, and for cathodically-treated water8-9. However, the patent further discloses that the apparatus proposedaims to achieve solutions of active chlorine with a pH of between 4.5and 7.5 used as a sterilizing solution, disinfectant, decontaminant,bleaching agent, detergent or medicine with antibacterial and antiviralaction.

K. S. Venkitanarayanan et al. (Appl. & Env. Microbiol., 65(9), pp.4276-4279, September 1999) evaluated the efficacy of electrolyzedoxidizing water for inactivating E. coli O157:H7, Salmonellaenteritidis, and Listeria monocytogenes. A five-strain mixture of E.coli O157:H7, S. enteritidis, or L. monocytogenes was inoculated inelectrolyzed oxidizing water at various temperatures, for various timeperiods. The electrolyzed oxidizing water is produced from a saline basesolution containing approximately 12% by weight NaCl. The electrolyzedoxidizing water is also described as having a 0.1% salt, Cl₂ of 10-80ppm, pH less than 2.7 as well as an electrolyzed oxidizing water havingCl₂ of 73-86 ppm, and pH of 2.38-2.48. At 4° C. and 23° C., an exposuretime of 5 minutes, the population of all three pathogens in thetreatment samples was reported to be reduced by approximately 7 logCFU/mL, with compete inactivation by 10 minutes of exposure. A reductionof greater than 7 log CFU/mL in the levels of the three pathogens wasreported to occur in the treatment samples incubated for 1 minute at 45°C. or for 2 minutes at 35° C.

SUMMARY OF THE INVENTION

This invention relates to stable physiologically balanced, non-cytotoxicionized, acidic solutions and to a methodology for their use. Theinvention also relates to applications of the solutions of theinvention, including a specialized bandage which may be used incombination with the solutions, or with other topically appliedmaterials. The ionized solutions may be prepared by electrolysis. Inanother aspect of the invention, the solutions are prepared by chemicalmethods, including synthesis, or by mechanical methods such as bymixing, or are prepared in situ.

A novel physiologically balanced solution was recently disclosed inco-pending applications, U.S. application Ser. No. 10/209,681, filedJul. 30, 2002, U.S. application Ser. No. 10/000,919, filed Nov. 2, 2001,and U.S. Ser. No. 09/482,159, filed on Jan. 12, 2000 (corresponding toWO 01/54704 A1 published on Aug. 2, 2001), all of which are incorporatedherein by reference in their entirety.

The composition of the invention may be prepared using an inorganic saltin physiologically balanced form. The inorganic salt is used in order tomimic the electrolyte concentration and mixture of extra cellular bodyfluid in an isotonic state. The solution typically comprises the halidesalt of sodium, or potassium, or calcium, and other cations. Typicallythe halide is fluoride, chloride, bromide, or iodide, and most typicallychloride. In part, the concentrations of the salinity, the pH and theactive chlorine concentration are such that they give the compositionits unique properties.

The solutions of the present invention may be prepared using a singleinorganic salt, forming an initial concentration of the salt in theaqueous solution of about 0.4 to about 1.0%. The halide-comprising saltmay be selected from the group consisting of lithium halide, sodiumhalide, potassium halide, magnesium halide, calcium halide, zinc halide,cesium halide, rubidium halide and barium halide. Non-limiting examplesof the inorganic salt may also include NaBr, NaI, NaF, LiBr, LiCl, LiI,MgI₂, MgBr₂, KI, KCl, KBr and the like. The inorganic salt may be ametal halide such as a chloride comprising salt selected from the groupconsisting of LiCl, NaCl, KCl, MgCl₂, CaCl₂, and ZnCl₂. In one aspect ofthe invention, the initial salt concentration used in the aqueoussolution is about 0.4 to about 0.9%.

In another aspect of the invention, the inorganic salt is sodiumchloride at a concentration of about 0.4 to about 1.0% NaCl which isabout four-tenth to slightly higher than full strength of normal orisotonic saline solution. According to Parker's McGraw-Hill Dictionaryof Scientific and Technical Terms, S. P. Parker, editor, Fifth Edition,“normal saline”, “physiological saline”, “physiological salt solution”are defined as a “solution of sodium chloride in purified water,containing 0.9 grams of sodium chloride in 100 milliliters; isotonicwith body fluids.” For different salts such as lithium halides,potassium halides, and the like, the concentration of the salt insolution making up an isotonic solution may differ from theconcentration of sodium chloride in an aqueous solution in order tomaintain the desired osmolarity of the solution of the invention. In yetanother aspect of the invention, the sodium chloride in the aqueoussolution is at a concentration of about 0.4 to about 0.9%.

In one aspect of the present invention, we have created a compositioncomprising a stable, physiologically balanced, noncytotoxic acidicsolution, herein also referred to as the NVC-101 solution, where thestarting solution prior to its preparation, for example, byelectrolysis, comprises a total concentration of the halide-comprisingsalt ranging from about 0.4 g/L to about 16 g/L; more preferably rangingfrom about 4 g/L to about 10 g/L; and, most preferably, ranging fromabout 4 g/L to about 9 g/L. The solution may optionally containminerals. The solution is adjusted to a pH within the range of about 2to about 5, and has an oxidation reduction potential within the range ofabout +600 mV to about +1200 mV, and the solution having a total activehalogen concentration of 0.1 to about 1,000 ppm, preferably from about10 to about 200 ppm, and most preferably from about 40 to about 190 ppm.In one aspect of the invention, the active halogen is selected from thegroup consisting of fluorine, chlorine, bromine, and iodine. In anotheraspect of the present invention, the halogen is chlorine.

The starting solution used to prepare the physiologically balanced,acidic composition of the invention may comprise a halide-comprisingsalt selected from the group consisting of lithium halide, sodiumhalide, potassium halide, magnesium halide, calcium halide, zinc halide,cesium halide, rubidium halide and barium halide. The composition of thesalts of the solution of the present invention are physiologicallybalanced, as salt contents that are too low or too high in concentrationrelative to a physiological balanced solution may damage cells. The term“starting solution” is defined as the solution containing the added saltcomposition prior to any reaction or electrolysis of the solution.

In another aspect of the invention, the starting solution of thehalide-comprising salt, and optionally containing minerals, is convertedto an acidic water solution through electrolysis. The electrolyzed,halide-comprising solution has a typical oxidation reduction potential(ORP) of about +600 to +1200 mV. The pH of the electrolyzed, halidecomprising solution, such as a chlorine-comprising solution, istypically lowered to about 5 or less, but not less than a pH of 2,preferably with a pH range of about 3.0 to 4.0, more preferably a pH ofabout 3.5 to 4.0, most preferably a pH of about 3.5, giving the solutionvirucidal, bactericidal, fungicidal, and sporicidal properties. Thehalide-comprising acidic solution is physiologically balanced. Typicallythe salts are supplied in the form of a halide-comprising salt which isionized during electrolysis. These physiologically-balancinghalide-comprising salts are selected from the group consisting oflithium halide, sodium halide, potassium halide, magnesium halide, zinchalide, lithium halide, barium halide, cesium halide, and rubidiumhalide. Preferably, these physiologically-balancing halide-comprisingsalts are selected from the group consisting of lithium halide, sodiumhalide, potassium halide, magnesium halide, zinc halide, lithium halide,and barium halide. Most preferably the salts are selected from sodiumchloride, potassium chloride, magnesium chloride, or zinc chloride.

In another aspect of the invention, the starting solution for thepreparation of the electrolyzed solution comprises of at least one metalhalide salt. Where more than one metal halide salts are present, thesalts may be present in the same or different concentrations from eachother.

In one exemplary solution of the present invention, the startingsolution for the preparation of the electrolyzed solution includessodium halide present at a concentration ranging from about 4.0 g/L toabout 9.9 g/L. In one aspect of the invention, the halide is chloride.

A particularly preferred starting solution for preparation of thesolution includes sodium chloride present at a concentration rangingfrom about 0.4 g/L to about 14 g/L.

In one aspect of the invention, the solution of the invention may beprepared by electrolysis by subjecting the starting salt solution toelectrolysis under conditions sufficient to produce the desiredcomposition.

In another aspect of the invention, the salt comprising acidic solutionsmay be prepared by chemical methods, including chemical synthesis, or byphysical methods such as mixing the components of the solution. Inanother aspect, the solution is prepared in situ at the location whereit is to be applied or used directly. Methods for the preparation of thesolution in situ are provided below.

The acidic solution of the invention contains hypohalous acid and maycontain, among other components, hydroxyl free radicals, oxygen, andozone. These components comprise some of the same oxidizing agentsinvolved in physiological systems associated with wound healing andtissue repair and regeneration. For example, hypochlorous acid is thechief bactericidal agent produced by neutrophils at sites ofinflammation, injury, and wounds.

Because the solutions of the invention are physiologically balanced,when applied to infected wounds, they enhance the process of healingsubstantially. Antimicrobial properties of the solutions of theinventions have been tested against many organisms, includingEscherichia coli, Listeria monocytogenes, Staphylococcus aureus,methicillin-resistant S. aureus (MRSA), Pseudomonas aeruginosa,Lactobacillus, yeast, vancomycin-resistant enterococcus, molds, andspores, including spores of anthrax. In particular, the solutions of thepresent invention have been used to successfully treat three differentstrains of Bacillus anthracis. Vancomycin-resistant bacteria, MRSA, andothers are easily destroyed by the solutions of the present invention.The solutions of the invention are osmotically balanced, environmentallyfriendly, and have minimal cytotoxicity. For example, no cytotoxicitywas observed in rabbits' eyes nor in in vitro cytotoxicity studiescarried out to date.

When the solution of invention is applied in in vitro studies to humanskin cells: keratinocytes, fibroblasts and melanocytes, it is welltolerated and the minimal-cytotoxicity parallels that of sterile salinesolution. The solution of invention was also applied in in vivo studiesto rabbit eyes using the Draize test, which provides direct observationsof the eyes' anatomical and physiological changes after exposure of theeyes to test solutions. In comparative studies, rabbits receivedrandomly and in a double-blind fashion either saline (15 eyes) or thesolutions of the present invention (15 eyes). Each eye received 0.1 mlof solution every 8 hours and observations were collected at varioustime points. The treated eyes were observed for ocular irritation. Thecytotoxicity index was zero for both arms of the studies: saline and thesolutions of the invention treated rabbits tolerated both treatmentssimilarly, and did not show any irritation response. The isotonicsolutions of the present invention were determined to be non-toxic tobiological tissues and comparable to saline solutions.

In one aspect, the solution of the invention has the following stabilitycharacteristics. After the solution is stored in a container or storagemedium for a period of about 25 months at about 4° C., the solution wasdetermined to have a measured oxidation reduction potential (ORP) of noless than about 90% but not more than about 99.9%, preferably no lessthan about 95% but no more than about 99.9%, and most preferably no lessthan about 97.5% but not more than about 99.9% of the ORP of thesolution freshly prepared prior to storage, while maintaining up to 5logs of reduction in the activity of the microorganisms after 10 to 60seconds of exposure to the solution.

The stable solutions prepared and stored in a medium according to themethods of the present invention have extended stability or shelf lifecharacteristics, depending on the nature of the medium of storage, thetemperature of storage, and whether the container or medium has beenopened. For example, the solution may have a ORP of no less than 95% ofthe ORP of the freshly prepared solution for at least 24 months whenstored at room temperature if the container has not been previouslyopened or used after storage. In one aspect, the stable solution of thepresent invention may be stored in a gas tight, sealed container whichfurther extends the stability characteristics of the solution. Inaddition, the solutions of the present invention will have a longerstorage shelf life if the solutions are stored below room temperaturerather than when stored at or above room temperature. “Room temperature”is being defined herein as between 20 to 25° C.

As defined herein, “stability” of the solution or a “stable solution”means that the solution of the present invention maintains up to 5 logsof reduction in the activity of the microorganisms after 10 to 60seconds of exposure to the solution.

The relative stability of the solution of the invention may also bedetermined from chemical analysis or by spectroscopy. The stability ofthe solution may be determined by iodometric titration or by UV-VIS forthe presence of active halide as described herein.

A stable solution as defined in this invention is a solution preparedand stored according to the procedures described herein and having areduced concentration of active chlorine over a period of time,preferably a reduction of between 1-95% of active chlorine species, morepreferably a reduction of between 5-15% of active chlorine species, andmost preferably a reduction of between 0-5% of active chlorine speciesin solution as determined by UV-VIS or iodometric titration over aperiod of at least one month, preferably at least 2 months and morepreferably, at least 3 months.

The measurement of the pH of a solution of the present invention isanother complementary method for determining the stability of thesolution in addition to the UV-VIS or iodometric titration method.

The concentration of “active chlorine” or “free chlorine” species asdefined herein, refers to chlorine comprising species such as HOCl,NaOCl and Cl₂ present in a solution of this invention, and the totalconcentration of all of the active chlorine or free chlorine species insolution can be determined by UV-VIS or by iodometric titration. Theactive chlorine species in solution may also be expressed as[HOCl]_(total) where [HOCl]_(total) is defined as the sum of theconcentration of HOCl, OCl⁻, and Cl₂ in solution; that is,[HOCl]_(total)=[HOCl]+[OCl⁻]+[Cl₂]. The concentration of active chlorinespecies may also be expressed in ppm, where the ppm concentration isequal to the number of mM of the species times the molecular weight ofthe particular species being measured. For example, if a concentrationof Cl₂ is referred to in ppm unit, conversion to the concentration in mMrequires that the concentration in ppm be divided by the molecularweight of Cl₂ (MW of 71).

Similarly, the concentration of the “active halogen” or “hypohalousacid” species refers to the concentration of the corresponding halogencontaining species HOX, NaOX, or X₂ as discussed above, where X is ahalogen atom.

The species HOCl, NaOCl and Cl₂ are equivalent in their reactions in theiodometric titration methods or as determined by the UV-VIS method asfollows:

HOCl+HCl

Cl₂+H₂O

HOCl+NaOH

NaOCl+H₂O

Cl₂+2NaOH

NaOCl+NaCl+H₂O

The active chlorine species in a solution may be measured by aniodometric titration method, by reacting the solution with KI and thentitrating the solution with a Na₂S₂O₃ solution in the presence ofstarch. The reaction of the active chlorine species occurs as follows:

HOCl+HCl+2KI

I₂+2KCl+H₂O

Cl₂+2KI

I₂+2KCl

2HCl+NaOCl+2KI

I₂+2KCl+NaCl+H₂O

I₂+2Na₂S₂O₃

NaI+Na₂S₄O₆

The active chlorine can also be reacted with NaOH and is converted toNaOCl.

NaOCl has an absorption at 292 nm with a known molar absorptivity of 362M⁻¹ cm⁻¹. Therefore, the concentration of the active chlorine speciescan be measured and is directly correlated with the concentration withany one of the species HOCl, NaOCl, or Cl₂ or with the combination ofthese species in a 1:1 basis.

Similarly, the concentration of a hypohalous acid species in solution asdefined herein correspond to the concentration of the active bromine orfree bromine, active iodine or free iodine, and active fluorine, or freefluorine species in the solution as defined above.

In one aspect, the solution of the invention has the following reducedcytotoxicity. When the solution of invention is applied in in vitrostudies to human skin cells such as keratinocytes, fibroblasts andmelanocytes, it is well tolerated and no substantial cytotoxicity wasmeasured using Tripan Blue intergen detection and pro check cellviability assay. In another aspect, the solution of the presentinvention exhibits the minimal-cytotoxicity parallels to that of sterilesaline solution.

Without being bound by any theory offered herein, it is believed thatthe minimal cytotoxicity of the solution of the present inventiondepends on the concentration of OCl⁻ in the solution as disclosedherein.

Because the composition of the present invention is nontoxic and hasantibacterial properties it is useful in any application in whichantimicrobial properties are desirable. Such applications include,without limitation, treatment of wounds, burns, and canker sores;irrigation; cleaning of tissue sites (e.g., pre- and post-operative);ophthalmic applications (e.g., in contact lens cleaning solutions or forirrigation of the eye before, during, or post ophthalmic surgery); fordermatological applications, psoriasis; and numerous applications whichare readily apparent to one skilled in the art. Unlike many otherinorganic halide solutions used in similar applications, the compositionof the invention has minimal to no side effects. For example, in Draizetesting in Rabbit eyes, when compared to other antiseptic solutions, thephysiologically balanced, stable, acidic solution of the presentinvention behaves in a manner similar to saline solution.

In another Draize test, rabbit's eyes were treated with the solution ofinvention and compared with the ophthalmic grade Betadine (manufacturedby: Alcon Co., TX, at a 5% concentration). Each eye received 0.1 ml ofsolution every 8 hours and observations were recorded at various timepoints. The Draize method relies on direct observations of the eyes'anatomical and physiological changes after exposure of the eyes to testsolutions. Rabbits treated with the solution of invention tolerated thetreatment without any signs of irritations, whereas, rabbits treatedwith ophthalmic grade Betadine did not tolerate the treatment and showedsignificant level of redness, ocular irritation and discomfort.

The composition of the invention can be incorporated into a variety ofapplications, including a bandage or wound dressing, as describedsubsequently herein. The physiologically balanced, acidic solution maybe used in combination with a specially designed bandage in a woundtreatment protocol as described subsequently herein. The specializedbandage includes an opening or “window” through which topical treatmentmaterials such as the solution of the present invention may be applied.

Also disclosed herein is an article of manufacture comprising thecomposition of the invention packaged in a container. Surfaces of thecontainer which are in contact with the composition of the invention aremade of material which is not reactive with an oxidizing agent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a cross-sectional schematic of an electrolyzing unit 1 havingtwo compartments, identified in FIG. 1 as elements 2 and 3. Compartments2 and 3 are separated by a semipermeable membrane 4. A positiveelectrode 5 is located in compartment 2, where a strong acidic solution6 is generated. A negative electrode 7 is located in compartment 3,where an alkaline solution 8 is generated. Electrodes 5 and 7 areconnected to a power source 9 which generates a current acrosssemipermeable membrane 4. A lid 10 keeps electrolyzing unit 1 free fromambient air 11.

FIG. 2A is a schematic top view of an air-permeable bandage 200,including outer portion 201 having a primary adhesive border 202; aninner portion 210 including a lifting flap 205 having a secondaryadhesive border 207, a lifting tab 204, which assists in the lifting offlap 205, a hinge 206, and a dew/humidity indicator 208 (or othersensor/indicator as will be described subsequently herein).

FIG. 2B is a schematic side view of air-permeable bandage 200, showinglifting flap 205 and lifting tab 204 in a partially lifted position, toprovide a window opening 203 through bandage 200. A portion of secondaryadhesive border 207 has been lifted above the upper surface 209 ofbandage 200.

FIG. 2C is a schematic cross-sectional view of air-permeable bandage200, with lifting flap 205 and lifting tab 204 in a lowered position,secured to upper surface 209 of bandage 200 by secondary adhesive border207.

FIG. 3 is a schematic cross-sectional view 300 of an air-permeablebandage 200 of the kind shown in FIGS. 2A-2C, applied over asubcutaneous wound 303. The subcutaneous tissue 304 is packed with gauze306 which has been soaked in the physiologically balanced, electrolyzed,acidic solution 308 of the present invention. The bandage 200 is adheredto the skin surface 302 by a primary adhesive border 202. Bandagelifting flap 205 can be lifted via tab 204 to expose gauze 306 for theapplication of additional solution 308 when a dew/humidity indicator(not shown) or other sensing/indication device (not shown) indicates alow level of humidity of the gauze 306.

FIG. 4 represents PFA in NVC-101 in torch-sealed glass ampules at 40° C.(Oven).

FIG. 5 represents a distribution of chlorine species vs pH in 0.9% NaCl.

FIG. 6 a represents a HOCl stability of NVC-101 JY-1-175 in torch-sealedclear glass ampules.

FIG. 6 a represents a pH stability of NVC-101 JY-1-175 in torch-sealedclear glass ampules.

DETAILED DESCRIPTION OF THE INVENTION

Described herein are stable, physiologically balanced, acidic solutions;methods and apparatus used in the production of the solution; methodsfor use of the solution, including the description of a specializedbandage for administering the solution or other topically appliedtreatment materials. Also disclosed are recommended packaging for thesolution.

I. The Composition of the Invention

The present invention is a physiologically balanced, acidic solution,which may be generated from a starting solution comprising a totalconcentration of one halide-comprising salt ranging in osmolarity fromabout 0.014 to 0.547 osmol; more preferably ranging from about 0.123 to0.376 osmol; and most preferably ranging from about 0.137 to 0.342osmol. Optionally, minerals may be added, depending on the end useapplication.

A typical starting solution, prior to electrolysis, by way of exampleand not by way of limitation, may comprise of one chloride comprisingsalt selected from the group consisting of lithium chloride, sodiumchloride, potassium chloride, magnesium chloride, calcium chloride, zincchloride, cesium chloride, rubidium chloride, and barium chloride.

Representative concentration ranges for the various chlorine-comprisingsalts that may be used in the starting solutions used to preparesolution are presented in Table 1, below.

TABLE 1 Compositions of Chloride Containing Salts In PreferredEmbodiment Starting Solutions For Preparation Of An Acidic SolutionPreferred More Preferred Most Preferred Solution Salt MW (g/mole) Ranges(g/L) Ranges (g/L) Ranges (g/L) 1 NaCl 58.50 0.400 to 16.000 3.600 to11.000 4.000 to 10.000 moles→ 0.007 to 0.274 0.062 to 0.188 0.068 to0.171 osmoles → 0.014 to 0.547 0.123 to 0.376 0.137 to 0.342 2 KCl 74.590.510 to 20.401 4.590 o 14.025 5.100 to 12.750 moles→ 0.007 to 0.2740.062 to 0.188 0.068 to 0.171 osmoles → 0.014 to 0.547 0.123 to 0.3760.137 to 0.342 3 MgCl₂ 95.30 0.434 to 17.377 3.910 to 11.946 4.344 to10.860 moles→ 0.005 to 0.182 0.041 to 0.125 0.046 to 0.114 osmoles→0.014 to 0.547 0.123 to 0.376 0.137 to 0.342

Definition of Osmolarity: A 1 M solution of a non-dissociable solute is1 Osmolar. (The solution contains 6.023×10E23 particles per liter). Thesolution of dissociable salt is n Osmolar, where n is the number of ionsproduced per molecules. Thus a 0.03 M solution of KCl is 0.06 Osmolar.(Irwin H. Segel, Biochemical Calculations, 2nd edition. Published byJohn Wiley & Sons, New York). Osmolarity is often considered inphysiological studies where tissue or cells must be bathed in a solutionof the same osmolarity as the cytoplasm in order to prevent the uptakeor release of water. Blood plasma is 0.308 Osmolar. Thus the red bloodcells suspended in a 0.308 Osmolar NaCl solution (0.154 M) would neithershrink nor swell. The 0.154 M NaCl solution is said to be isotonic withrespect to the red blood cells (Irwin H. Segel et al).

The properties of the physiologically balanced, acidic solutionsproduced from the Starting Solutions described in Table 1 are presentedin Table 2, below.

TABLE 2 Properties of Preferred Physiologically-Balanced AcidicSolutions Generated From NaCl Starting Solution Listed in Table 1Preferred More Preferred Most Preferred ORP (mV) +600 to +1200 +800 to+1190 +1000 to +1180 pH 2.0-6.0 2.2-5.5 2.4-5.0 Hypochlorous 0.1-1000  1-200  60-190 Acid Conc.(ppm) Molar Ratio¹ about 0-2.55 about 0-0.82about 0-0.26 range of OCl⁻ over sum of OCl— and HOCl at 20° C. (%) ¹Geo.Clifford White: Handbook of Chlorination and Alternative Disinfectants,page 218, 4^(th) ed., John Wiley & Sons, Inc. New York, 1999.

II. Apparatus and Method for Making the Physiologically Balanced,Electrolyzed, Acidic Wound Healing Solutions

The physiologically-balanced, acidic solution of the invention may beprepared using electrolysis. Electrolysis of water is the process bywhich the hydrogen ions are reduced, providing hydrogen gas, and thehydroxide ions are oxidized, providing oxygen gas.

The wound healing solution described herein was prepared using aSUNTRON® MWB-2 model electrolyzing unit of the kind manufactured byKoshin Co. Ltd., Kyoto, Japan. Equivalent wound healing solutions can beprepared using a SUPER OXSEED LABO® electrolyzing unit of the kindmanufactured by ARV Co., Japan.

With reference to FIG. 1, which shows a general schematic of anelectrolyzing unit in which a physiologically balanced, electrolyzed,acidic wound healing solution is prepared, and with reference to theSUNTRON® MWB-2 model electrolyzer, the electrolyzing unit 1 has a firstcompartment 2 and a second compartment 3, each of which have a capacityof about 3 liters. Compartments 2 and 3 are separated by asemi-permeable membrane 4. In the first compartment 2, a positiveelectrode 5 is located. In the first compartment 2 a strong acidicsolution 6 is generated. In the second compartment 3, a negativeelectrode 7 is located. In the compartment 3, an alkaline solution 8 isgenerated. Electrodes 5 and 7 are connected to a power source 9 whichgenerates a 0.9 A, 100V current. A lid 10 keeps the electrolysis unitfree from contamination by ambient air 11.

13.5 g of Sodium chloride (Non-iodated, Morton) was added to 2.5 litersof distilled water to form a 5.38 g/liter or a 0.538% solution. 2.5 L ofthe solution was placed in first compartment 2 and 2.5 L of solution wasplaced in second Compartment 3. The power source 9, shown in FIG. 1, wasturned on and power was applied for 15 minutes. The electrolysis wascarried out at room temperature (about 25° C. to 30° C.), with noexternal heat added and no heat removed.

Salt solutions allow currents to pass between the electrodes,accelerating the process of electrolysis. The amount of salt necessaryto affect the electrolysis process is minimal. During the electrolysisprocess, a halide salt, such as sodium chloride is in ionized form, asshown below.

During electrolysis of saline, the sodium ions are attracted to thenegatively charged electrodes, and will counterbalance the hydroxideions on the alkaline side; the chloride ions travel to the positiveelectrode. The chloride ions then undergo an oxidative process whichresults in the generation of small quantities of chlorine gas that areimmediately consumed to form hypochlorous acid, as illustrated below.

Chlorine species are in the form of HClO, ClO⁻, or Cl⁻; the balanceamong these ions is greatly affected by the pH of the solution. Withoutbeing bound by any theory, it is believed that HClO and ClO⁻ ions areeffective sterilizing agents, with HClO being ten times more effectivethan ClO⁻. In acidic pH, most of the ClO⁻ ions are in the form of HClO.

Other halide salts undergoing electrolysis participate in similarionization processes are well known and documented in the art.

An example of a typical physiological-balanced acidic solution of theinvention has a concentration of sodium chloride ranging from about 0.5to 9.9 g/L.

In one aspect of the invention, the concentration of hypohalous acid(HOX) in the solution is from about 0.1 to about 1,000 ppm, morepreferably from about 1 to about 750 ppm, and most preferably from about5 to about 500 ppm.

In one aspect of the invention the physiologically-balanced,electrolyzed acidic solution of the invention has a concentration ofsodium cations ranging from about 0.01 g/L to about 7 g/L.

A typical physiologically-balanced, electrolyzed acidic solutionproduced using the starting materials described the invention has a lowpH (about 2 to about 5), and an HClO concentration of about 0.1 ppm toabout 1000 ppm. In one aspect of the invention, the pH range of thesolution is 2.4 to 5.0. This combination of chemicals gives theelectrolyzed acidic saline solution of the invention its superiorantiseptic ability and its extended stability properties. In addition,the solution is characterized by remaining stable and active when storedfor at least three months at room temperature.

A typical physiologically balanced solution of the invention ischaracterized by an oxidation reduction potential (ORP) from about +600mV to about +1200 mV.

Standard electrolysis equipment, including the particular apparatusnamed herein, can be used in the manufacture of the electrolyzed saltsolutions of the invention, as previously mentioned.

III. Chemical Processes for Preparing the Physiologically Balanced,Acidic Solutions

Various chemical processes for the preparation of aqueous solution ofhypochlorous acid are known in the art. For example, see The MerckIndex, Tenth Edition M. Windholz, Ed., Merck & Co., Rahway, USA, 1983and references cited therein. More generally, non-limiting examples ofprocesses for the preparation of the solution of the present inventionare provided in the following Reaction Scheme:

Non-limiting examples of processes for the preparation of the aqueoussolutions of the present invention are provided as follows:

NaOCl+HCl→HOCl+NaCl

NaOCl+NaHSO₄→HOCl+Na₂SO₄

Ca(OCl)₂+2HCl→2HOCl+CaCl₂

Ca(OCl)₂+2NaHSO₄→2HOCl+CaSO₄

Na₂SO₄

In each of the above representative processes for the preparation of thesolution, upon the formation of the desired solution of the invention,the pH of the solution may be adjusted to the desired pH using standardmethods known in the art for adjusting the pH of aqueous solutions.

In one aspect of the invention, the relative concentrations of reactantsthat will yield the composition of the stable aqueous physiologicallybalanced solutions of the present invention will vary according to thenature and type of reactants used to form the desired solutions. Forexample, the concentration of the salts comprising the solution of theinvention may include the concentration ranges as disclosed in Table 1.

In one aspect of the invention, the stable aqueous physiologicallybalanced solution of the present invention may also be prepared by themixing of the appropriate starting chemicals immediately before usingthe solution.

In another aspect of the invention, the stable aqueous physiologicallybalanced solution of the present invention may also be prepared in situby mixing the chemicals immediately before use. In situ mixing of thestarting materials may be performed using various known methods in theart. For example, starting materials or reagents for the preparation ofthe composition of the invention may be separately stored, encased orcontained in glass beads, ampules and the like, and the reagents can beadmixed when the individual containers or beads encasing the reagentsare released and allowed to react at the desired site for applying thesolution. Where the reagents are contained in glass beads, ampules orthe like, means for binding or holding the individual containerstogether, while allowing the release of the reactive components of thesolution, may be accomplished to prevent the release of the containersat the desired treatment site.

Following manufacture, the solutions of the invention must be stored foruse. Methods and materials of packaging are very important inmaintaining and extending the useful shelf life of the solutions. Forexample, the surfaces of the containers which make contact with thesolution should be made of a material which tends not to react withoxidizing agents.

We evaluated a number of different container materials, and surprisinglydiscovered that while a glass contacting surface preserves the long termstrength (potency) of the solution, certain plastic surfaces ormaterials are not compatible with the solution for long term storage. Byway of example and not by way of limitation, chemically resistant,coated soda lime amber glass 1 L or 500 mL bottles (manufactured byLawson Mardon Wheaton, Millville, N.J. 08332), meeting the requirementsfor Type III as established by the United States Pharmacopoeia, VolumeXXIII (1995), and supplements thereto, under “Chapter <661>, ChemicalResistance-Glass Containers” make excellent storage containers for thephysiologically-balanced solutions of the present invention. Thesebottles also meet the requirements for light protection established bythe USP under Chapter <661>, “Light Transmission”, which may be helpfulin some instances. The bottle cap is fabricated from phenolic, and has aliner facing made out of TEFLON® (PTFE) which is less reactive thanphenolic, and which helps seal the cap, preventing the passage ofambient air into the bottle. This bottle is available from AllPak Corp.,Bridgeville, Pa.

A white (clear) glass bottle produced by the same manufacturer (AllPakCorp.), but absent the amber coloring also functions well in maintainingthe stability of the solution. In one aspect of the invention, a gastight sealing of the solution storage container preserves or extends thestability characteristics of the solution. Gas tight sealing methodsemployed for the storage, sealing and resealing of the containers afterused may include methods known in the art such as using air tight screwcaps, air tight lids, caps or lids having chemical resistant O-rings orgaskets, the use of sealing tapes, such as electrical tapes, or relatedmethods known in the art for the airtight sealing or resealing ofcontainers.

Cl₂ and HOCl are known as strong oxidants, and these species are knownto react with many different synthetic plastic materials. An appropriatenon-glass packaging material for the NVC-101 solution should benon-reactive to both Cl₂ and HOCl, and also not permeable to the speciesof the NVC-101 solution. We have studied the stability of HOCl at pH 3.5in PET bottle, Barex bottle (British Petroleum), LDPE bottle, highdensity polyethylene bottle, polypropylene bottle, PFA bottle (SavilleCorporation), Eastman Plastics bottle, PVC bottle and Pouch (white poly,foil, special high chemical resistant adhesive). In order to test ifthere are reactions of the materials with HOCl or Cl₂, the materialswere cut into small pieces (about 1 gram strips) and sealed in the 30 mLampules with the HOCl solutions (about 25 ml) at pH 3.5. The resultsshow that among all of the above materials tested, PFA, a type of Teflonmaterial, is the only material tested that does not react with HOCl orCl₂ in the NVC-101 solution under these conditions (FIG. 4). Therefore,PFA can be used as the packaging material for the NVC-101 solution.

As defined herein, container materials or compositions that may be usedfor the storage of the NVC-101 solutions are considered to be“non-reactive”, “does not react”, “not permeable”, are “chemicallynon-reactive”, or are “resistant to oxidative degradation” are materialsthat are resistant to react with the NVC-101 solution and allow longterm storage of the solution without adversely affecting the stabilityof the solution.

TABLE 3 Preparation of Solution 1 in Table 1 using a Synthetic Method(0.9% salt solution)^(a) MW Weight Volume Molarity Reagent (g/mole) (g)mmoles (mL) (moles/liter) H₂O 18 ~494.7 NaCl 4.34 NaOCl 74.5 1.8 1.31.60 0.805 HCl 36.5 3.7 3.7 3.7 1 ^(a)See the Procedure #1 for thepreparation of the 0.9% salt solution.

Stability of Hypochlorous Acid in 0.9% Saline Solution:

The decomposition of a mixture of hypochlorous acid and sodiumhypochlorite was studied by Chapin from pH 1 to pH 13 in 1934. Chapin,R. M. J. Am. Chem. Soc. 1934, 56, 2211-2215. Chapin found a maximumdecomposition rate in the neutral pH range. The following stoichiometryand rate expression was found to approximate the experimentalobservations:

2HOCl+OCl⁻→ClO₃ ⁻+2H⁺+2Cl⁻  Eq. 1

-d[HOCl]/3dt=k[HOCl]²[OCl⁻]  Eq. 2

The decomposition was also studied by Yokoyama and Takayasu in theneutral pH range. See Yokoyama, T.; Takayasu, O. Kogyo Kagaku Zasshi1967, 70, 1619-1624. Their work was carried out in 0.8-4.6 M chlorideion to control the ionic strength, and no buffer was used. The authorsproposed Eq. 3 as the rate expression based on their results, where aand b are constants.

—d[HOCl]/3dt=a[HOCl]²[OCl⁻]/(1+b[OCl⁻])  Eq. 3

A more detailed study of the decomposition of hypochlorous acid from pH5.0 to pH 8.0 was reported by Adam and co-workers. See Adam, L. C.;Fabian, I.; Suzuki, I., Gordon, G. Inorg. Chem. 1992, 31, 3534-3541.Under these conditions, they found that hypochlorous acid has a maximumdecomposition rate at pH 6.89. The overall stoichiometry ofdecomposition of hypochlorous acid and hypochlorite ion (HOCl+OCl⁻) inthe neutral pH region was determined as shown in Eq. 4.

xHOCl+(3−x)OCl⁻⇄ClO₃ ⁻+2Cl⁻ +xH⁺  Eq. 4

A mechanism (Eq. 5-10) for the decomposition was proposed by theseauthors, in which Cl₂O.H₂O is formed as an intermediate.

2HOCl⇄Cl₂O.H₂O  Eq. 5

OCl⁻+Cl₂O.H₂O→HOCl+HCl₂O₂ ⁻ k₂=3.0 M⁻¹s⁻¹ (50° C.)  Eq. 6

HCl₂O₂⇄HClO₂+Cl⁻  Eq. 7

HOCl+Cl₂O.H₂O→HOCl+H₂Cl₂O₂ k₂=3.6×10⁻³ M^(−b 1)s⁻¹ (50° C.)  Eq. 8

H₂Cl₂O₂⇄HClO₂+Cl⁻+H⁺  Eq. 9

HOCl+HCl⇄Cl₂+H₂O  Eq. 10

Above pH 6, the step in Eq. 6 is the rate determining step. Below pH 6,Eq. 8 becomes the rate determining step. This step is very slow. The k₂and k₂ values determined by Adam et al show that the reaction ofCl₂O.H₂O with OCl⁻ is nearly 1000 times faster than with HOCl,suggesting that HOCl is much more stable in the acidic condition than inthe neutral pH region. However, as the pH becomes acidic (pH<3), HOCl isconverted rapidly to Cl₂ in the present of Cl⁻ (Eq. 10), which isgenerated from HOCl self-decomposition (Eq. 5-10).

Synthesis of HOCl: In general, HOCl solutions were prepared byacidifying a NaOCl solution with HCl. The concentration of HOCl wasdetermined by either Iodometric titration with Hatch solution or UV-VISmethod. For analysis by UV-VIS method, aliquot of HOCl solution wasconverted to OCl⁻ (=362 M⁻¹ cm⁻¹ @ 292 nm; see Furman, C. F.; Margerum,D. W. Inorg. Chem. 1998, 37, 4321) with 0.1 M NaOH solution. A BeckmanpH meter was used to measure the pH of the solution.Distribution of Chlorine Species vs. pH in 0.9% Saline: The studies ofChapin, R. M., Yokoyama, T.; Takayasu, O., and Adam, L. C. et al showthat the decomposition of HOCl to give inorganic ions ClO₃ ⁻ and Cl⁻ isslow in acidic condition. However, decomposition of HOCl may also occurvia the formation of Cl₂ gas in the presence of excess NaCl (Adam, L. C.et al, 1992). The NVC-101 solutions as prepared according to a proceduredescribed herein contains HOCl, 0.9% NaCl (0.155 M), and has a pH ofabout 3.5. Equations 11-14 show the equilibria existing in NVC-101solution.

HOCl⇄H⁺+OCl⁻ pKa=7.5  Eq. 11

HOCl+C⁻+H⁺⇄Cl₂(aq)+H₂O K₁=9.6×10² M⁻²  Eq. 12

Cl₂(aq)+Cl⁻⇄Cl₃ ⁻⇄K₂=0.18 M⁻¹  Eq. 13

Cl₂(aq)⇄Cl₂(g) K₃=10.87 atm M⁻¹  Eq. 14

The pKa of HOCl is 7.5. See Gerrisen, C. M.; Margerum, D, W. Inorg.Chem. 1990, 29, 2758-2762. At pH 3.5, hypochlorite exists predominantlyas its conjugate acid form. At pH 7.5, [HOCl]/[OCl⁻]=1:1. As shown inEq. 12, high acidity (or higher concentrations of H⁺) favors theformation of Cl₂. See Wang, T. X.; Margerum, D. W. Inorg. Chem. 1994,33, 1050-1055. As the concentration of Cl₂ is increased in the solution,the formed Cl₂ may equilibrate to gaseous chlorine and is liberated tothe headspace of a container (Eq. 14). Therefore, degassing of Cl₂ tothe head space may become a major path for decreasing the concentrationof HOCl at low pH in 0.9% NaCl solution in storage containers with largea headspace.

The distribution of chlorine species over a pH range in a 0.9% NaCl isshown in FIG. 5. The Figure shows the specific pH ranges wherein Cl₂concentrations may be minimized. The chlorine species distributioncurves (% vs pH) in FIG. 5 were calculated based on the pKa, K₁, K₂ andK₃ values available from literature. See Gerrisen, C. M.; Margerum, D,W. Inorg. Chem. 1990, 29, 2758-2762; Wang, T. X.; Margerum, D. W. Inorg.Chem. 1994, 33, 1050-1055; Wang, T. X.; Kelly, M. D.; Cooper, J. N.;Beckwith, R. C. Margerum, D. W. Inorg. Chem. 1994, 33, 5872-5878; andBartlett, W. B.; Margerum, D. W. Environ. Sci. Technol. 1999, 33,3410-3414. The calculation results were verified by experiments. Theexperiments were carried out by using multiple wavelengths method, inwhich the molar absorptivities of HOCl, Cl₂(aq), and Cl₃ ⁻ are atwavelengths 220, 232, and 325 nm. The absorbances at these wavelengthswere used to set three equations according to Eq. 15:

A=ε ^(HOCl)[HOCl]+ε^(Cl3−)[Cl₃ ⁻]+ε^(Cl2)[Cl₂]  Eq. 15

The molar absorptivities used in this study are listed in Table 4.

TABLE 4 Wavelength ε^(HOCl) ε^(Cl2) ε^(Cl3-) nm M⁻¹ cm⁻¹ M⁻¹ cm⁻¹ M⁻¹cm⁻¹ 220 69.89 57 1.04 × 10⁴ 232 100 0 8800 325 10.8 70 180

Solving these equations give the concentrations of the chlorine species.The experimental results are also shown in FIG. 5. As shown in FIG. 5,appreciable amount of Cl₂ begins to form at pH<3.5. In order to minimizethe formation of Cl₂ gas, the pH of the solution should be controlled at3.5 or higher.

NVC-101 Solution Stability Experiments: The stability experiments werefocused on measuring the total concentration of the active chlorinespecies. The experiments were carried out in ampules at 40° C., roomtemperature and 4° C. with an initial total active chlorine speciesconcentration of 100 ppm (i.e. [HOCl]_(total)=100 ppm). The plots of[HOCl]_(total) vs Time and pH vs time in FIG. 6 show that the HOCl atthree temperatures is stable as determined by both [HOCl]_(total) and bythe pH of the solution. An initial drop in [HOCl]_(total) is observedwith samples taken within the first 24-48 hours. This initial drop maybe attributed to the loss of Cl₂ from the solution into the headspace.When the first data point was measured, the equilibrium as expressed inEq. 14 had not been established. By the time the ampule was opened foranalyzing the second data point after about 48 hours, the equilibrium inEq. 14 was reached. Compared to the first data point, more Cl₂ in thesolution had gone into the headspace of the ampule. After the initialdrop in [HOCl]_(total), no appreciable loss of HOCl was observed over180 days. This experiment shows that HOCl is stable at pH 3.5 in a 0.9%NaCl solution after the equilibrium distribution of Cl₂ in solution andthe gas phase was reached after about 48 hours.

Effect of Storage on the pH and Orp of the Solution in Screw CapBottles:

We conducted a study of the shelf life of the solution describedaccording to the composition of Table 3 to determine the effect ofextended storage in bottles made from various materials, and on the pHand oxidation-reduction potential (ORP) of the solution. Freshlyprepared solution was stored over a period of 3 months in 4 types ofscrew cap bottle: The amber glass; the white (clear) glass; High DensityPolyEthylene (HDPE); and TEFLON®. A variety of chemically non-reactivescrew caps and liners were also tested. The stability and activity ofthe solution of the present invention may also be measured bydetermining the active halogen concentration using UV-VIS spectroscopy.For solutions containing active chlorine, the stability and activity ofthe solution of the present invention may also be measured bydetermining the active chlorine concentration using iodometric titrationor using UV-VIS spectroscopy.

At given times over intervals of 5 to 10 days, known aliquots werewithdrawn to measure the pH and ORP. Thirteen aliquots were taken overthe testing period and each aliquots were measured for the pH and theORP. At a starting pH of 2.8, the solutions stored in amber glass, whiteglass, HDPE, and in Teflon maintained a pH of 2.8 over a period of morethan 75 days without change. It was also determined that containersusing a Teflon liner that is backed by soft silicone was most effectivein preventing the degassing of Cl₂ from the containers.

At a starting ORP value of 1175-1180, the solutions stored in amberglass, white glass, HDPE, and in Teflon maintained an ORP between 1150and 1175 over a period of more than 75 days without a significantreduction in the ORP.

Stability of NVC-101 Solution in Sealed Ampules:

20 mL of the NVC-101 solutions prepared according to Procedure #1 wasadded via a dispenser to 25 mL glass ampules. The glass ampules weresealed immediately using a torch. The initial total concentration ofactive chlorine species of the solution was 2.69 mM (i.e.[HOCl]_(total)=2.69 mM) as measured by iodometric titration.

The ampules were stored at 40° C., at room temperature, and at 4° C.Individual ampules stored at the different temperatures were openedafter 11 day, 15 days, 20 days, 29 days, 46 days, 62 days, 75 days, 103days, 127 days, 180 days and analyzed for the total concentration ofactive chlorine species by iodometric titration. In addition, the pH ofthe solution was measured using a freshly calibrated Beckman pH meter.

A graphical plot (FIG. 6( a)) of the [Chlorine]_(ppm) vs. time (in days)shows that the NVC-101 solutions at a pH of about 3.5 in a 0.9% NaClsolution prepared according to the methods described herein and storedin unreactive, sealed containers were stable at 40° C., at roomtemperature, and at 4° C. over a period of 180 days.

A graphical plot (FIG. 6( b)) of the pH of the solution vs. time (indays) shows that the NVC-101 solutions at an initial pH of about 3.5 ina 0.9% NaCl solution prepared according to the methods described hereinand stored in unreactive, sealed containers were stable at 40° C., atroom temperature, and at 4° C. over a period of 180 days.

The stability of a solution of this invention was investigated usingdifferent forms of packaging that would be practical for use by patients(Table 5). Sample A below represents the solution packaged in ninesingle-use 30 ml amber glass bottles with Teflon-lined screw caps andsealed with tape to ensure gas tightness. Sample B represents the samesolution packaged in a 250 ml amber glass bottle and Sample C representsthe same solution packaged in a 250 ml plastic bottle.

At the beginning of the experiment the concentration of free chlorinewas measured. Each day (except for two weekend days) the followingprocedure was employed.

-   -   1. At the beginning of the day, the 250 ml bottles were opened        for a period of two minutes and then closed.    -   2. At the end of the day, the 250 ml bottles were opened, a 20        ml sample was withdrawn and the bottle was closed after two        minutes. The 20 ml samples were tested for free chlorine        concentration.    -   3. At the end of the day one of the 30 ml bottles was opened and        tested for free chlorine concentration. The bottle was then        discarded.

TABLE 5 Stability of Solution over time in opened and closed containers[HOCl]_(total) in ppm^(a) Closed container Opened container Openedcontainer Day Sample A Sample B Sample C 0 184 184 184 1 184 171 150 2172 145 121 3 181 128 103 4 — — — 5 — — — 6 180 110 66 7 182 98 54 8 18059 38 9 177 49 31 10 179 42 27 ^(a)[HOCl]_(total) in ppm with a 5-10%experimental error.

The opening of the 250 ml bottles twice a day for two minutes wasdesigned to reflect the pattern of usage of a normal patient, where thepatient or a health care giver would be changing the dressing on thepatient's wounds and applying the solution twice a day.

It was surprisingly observed that the concentration of free chlorine andthus of hypochlorous acid was reduced very significantly over the periodof the experiment when the larger bottles were repeatedly opened asdescribed, whereas the single use bottles (30 ml) (Sample A in Table 5)maintained their concentration within acceptable levels. This indicatesthat each application of the solution of this invention should be from acontainer that has not been opened multiple times and preferably from asingle-use container, such as a bottle.

In one aspect, the solutions of the present invention may be stored insingle-use containers. In another aspect, the solutions of the inventionmay be stored in single-use containers of various different sizes,configurations, and having different volumes as suitable for the desiredapplications as disclosed herein. In some applications, for example, thesolution of the invention may be stored in single-use 30 mL, optionallydisposable containers.

Preparation of HOCl Solutions: in General, HOCl Solutions were Preparedby acidifying the NaOCl solution with HCl. The concentration of HOCl wasdetermined by either Iodometric titration with Hatch solution or UV-VISmethod. For analysis by UV-VIS method, aliquot of HOCl solution wasconverted to OCl⁻ (ε=362 M⁻¹ cm⁻¹ @ 292 nm; see Furman, C. F.; Margerum,D. W. Inorg. Chem. 1998, 37, 4321) with 0.1 M NaOH solution. A BeckmanpH meter was used to measure the pH of the solution.Reagents. All solutions were made with Millipore water, which wasvalidated with HPLC grade water. NaOCl (6%) solution was purchased fromVWR. NaCl and HCl used are reagent-grade.

Procedure #1: Preparation of NVC-101 Solutions

In a 500 mL Erlenmyer flask was placed NaCl (4.344 g). To this was added450 mL of distilled water, followed by 1.6 ml of 0.6% NaOCl (VWRInternational), and 3.7 mL of 1 Molar hydrochloric acid. This solutionwas transferred to a 500 mL volumetric flask and then enough distilledwater was added to reach the 500 mL mark. ORP, pH, and total availablechlorine were measured and recorded.

If sufficient acid is initially present in the solution to obtain thedesired pH range, then no pH adjustment is needed. Otherwise, the pH maybe adjusted to the desired range using standard methods known in the artfor increasing or decreasing the pH of the aqueous solution.

In one example, when the physiologically-balanced, acidic solution ofthe invention is stored in a glass bottle, the composition has beenshown to be stable for at least 90 days at room temperature.

Procedure #2: Preparation of 50 Liters of 1.70 mM HOCl Solution in 0.9%Saline at pH 3.5

The HOCl solution (1.70 mM) in 0.9% NaCl at pH 3.5 have been prepared inlarge scale using a 50-liter polyolefin plastic container as a reactionvessel. Table 6 lists the reagents and their quantity that may be usedto obtain the desired concentration and pH value.

TABLE 6 MW Molarity (g/ Weight Volume (moles/ Reagent mole) (g) mmoles(L) liter) Remarks H₂O 18 49.8 NaCl 58.5 440 440 g makes a 0.9% solutionin total volume of 50 liters NaOCl 74.5 92 1.70 0.98 HCl 36.5 2.5 0.1241.0

In order to avoid losing active chlorine during the manufacturing of thesolution, NaOCl was added in the last. An example of the manufacturingprocedure for an HOCl solution in 0.9% saline at pH=3.5 is described asfollows:

-   -   1. 440 g of NaCl was weighed in a 1000 mL-beaker, and then 500        mL Millipore water was added into the beaker to dissolve NaCl.    -   2. The solution was stirred for 5 minutes using a glass stir.        About half of the NaCl remained undissolved. The NaCl solution        was transferred into a 1000 mL volumetric flask. The undissolved        NaCl solid was retained in the beaker.    -   3. The 1000 mL volumetric flask was filled with Millipore water        up to the 1000 mL mark and the NaCl solution in the 1000 mL        volumetric flask was transferred into the vessel.    -   4. Another 500 mL Millipore water was added into the 1000 mL        beaker to dissolve the remaining NaCl solid. The solution was        stirred until all NaCl dissolved. Repeat step 3.    -   5. 124 mL of 1.0 M HCl was pipetted into the 1000 mL volumetric        flask. The flask was filled with Millipore water up to the 1000        mL mark. The HCl solution in the 1000 mL volumetric flask was        transferred into the vessel.    -   6. The vessel was filled with Millipore water up to 47 liters,        and the solution was stirred while the Millipore water was        added.    -   7. 92 grams of NaOCl (6%) was weighed into a 500 mL beaker and        the transferred into a 1000 mL volumetric flask and the flask        was filled with Millipore water up to 1000 mL mark. The solution        was transferred into the vessel. The volumetric flask was rinsed        and filled up to the 1000 mL mark with Millipore water. The        solution was transferred into the vessel.    -   8. The vessel was filled up to 50 liter.    -   9. The solution was stirred slowly and gently with a long glass        stir bar for about 2 minutes.    -   10. After about 2 minutes, a test for the active chlorine        concentration and pH of solution using UV-VIS method and pH        meter, respectively, was made to determine if the solution has        the desired concentration and pH.    -   11. The vessel was tightly capped. The solution was allowed to        sit in the vessel for about 2 hours to reach equilibrium.    -   12. The concentration of HOCl and pH of solution are analyzed on        UV-VIS spectrophotometer and pH meter before the solutions are        added to containers such as ampules or bottles.

Using the manufacturing methods described above, the desired freechlorine concentration (1.70±0.05 mM) and pH (3.55±0.05) areconsistently obtained.

An iodometric titration or UV-VIS method was used to determine theconcentration of active chlorine species in solution.

Iodometric Titration:

In an iodometric titration, the following reactions take place:

KI+HOCl→I₂+KCl

KI+Cl₂→I₂+KCl

KI+OCl⁻→I₂+KCl

I₂+starch (used as an indicator)→Blue complex

I₂+2S₂O₃ ²⁻→S₄O₆ ²⁻+2I⁻ (end point is colorless)

The total concentration may be determined as[HOCl]_(total)=[HOCl]+[OCl⁻]+[Cl₂]

Procedure for Iodometric Titration for Active Chlorine:

1. Set up titration apparatus using a HACH TetraStir™ apparatus asdescribed by HACH Digital Titrator Manual.2. Start with a clean 125-ml Erlenmeyer flask with a small stir bar inthe flask.3. Add the contents of 1 foil packet of Potassium Iodide Reagent(purchased from HACH) and one Dissolved Oxygen Powder Pillow (purchasedfrom HACH) to flask.4. Add Millipore water to the flask up to the 50-ml mark. Place flask onstir plate to completely dissolve the reagents.5. Add 5 ml of sample test solution to the flask using an acclimated5-ml pipet. Solution will turn yellow.6. Ensure that there is sodium thiosulfate solution throughout thedelivery tube, the counter is at zero, and the tip of the delivery tubeis dry. Then begin titration.7. Add sodium thiosulfate until the solution turns a very pale yellow.Then add 4-5 drops of starch indicator. The solution will turn blue.8. Continue titration with sodium thiosulfate until the solution turnsclear and remains clear for 30 seconds.

Using the above procedure for the analysis of NVC-101 solution inampules, each data point reported is the average concentration ofsolutions analyzed from three ampules.

UV-VIS Method:

The solution to be tested is basified before it is measured by UV-VIS.Basification converts both HOCl and Cl₂ into a same species, OCl⁻. OCl⁻has a strong absorbance at 292 nm, and therefore can provide a bettersignal and enhance the detecting limit.

Analysis using the UV-VIS method, the total concentration of the activechlorine species may be determined as[HOCl]_(total)=[HOCl]+[Cl₂]+[OCl⁻].

HOCl+NaOH→NaOCl+H₂O

Cl₂+2NaOH→NaOCl+NaCl+H₂O

Procedure for Measurement of Active Chlorine by UV-VIS:

1. Pipet 10 ml of 0.1 M NaOH solution into a clean and dry 50-ml beaker.2. Add 5 ml of NVC-101 test solution to this beaker. Mix with the NaOHsolution well.3. Use the basified NVC-101 solution to rinse the cell three times.4. Fill the cell with basified NVC-101 solution and cap the cell.5. Measure the OCl⁻ absorbance at 292 nm.6. The total chlorine concentration may be calculated using thefollowing equation:

[HOCl]_(Total)=3*(A _(292nm)/362)

where “3” is the dilution factor when the solution is basified, and “362M⁻¹ cm⁻¹” is the molar absorption of OCl⁻ at 292 nm.

Using the above procedure for the analysis of NVC-101 solution inampules, each data point is the average concentration of the solutionsfrom three ampules.

Antimicrobial Activity

Antimicrobial efficacy of a solution of the invention containing 9 g/LNaCl, 170 ppm hypohalous acid, having a pH of 3.0 and an ORP of 1175 wastested against microorganisms including Candida albicans, spergillusniger, Streptococcos pnemonea, MRSA, VRE, Baccilus subtillis, Bacillisceruis, Baccilus thorangensis, Baccilus anthracis, Pseudomonasaeruginosa, Escherichia coli, Staphylococcus aureus, Listeriamonocytogenes 10403s wild type, catalase-deficient mutant L.monocytogenes LM1370, Aspergillus niger (spores), Penecillium oblatum(spores), Lactobacillus, and E. coli 0157:H7. Up to 5 logs of reductionin the activity of the microorganisms was achieved after 10 to 60seconds of exposure to the solution of the present invention.

Antimicrobial properties: The solution of invention was effective in thetreatment of all microorganisms, including gram positive, gram negative,yeast, fungi and spore forming Bacillus, including different strains ofBacillus anthracis. The solution was found to exert pronouncedantibacterial action against all the microorganisms tested.

Eve and Skin Irritation Eve Irritation Experiment

The solution of the invention was evaluated for primary ocularirritation based on the requirements of the International Organizationfor Standardization 10993: Biological Evaluation of Medical Devices,Part 10: Tests for Irritation and Sensitization.

A 0.1 ml dose of the solution of the invention was instilled into thelower conjunctiva sac of the right eye of the screen rabbit and the lidwas gently closed for 1 second. The opposite eye was dosed with 0.1 mlof 0.9% sodium chloride (USP) as per sponsor to serve as the comparativecontrol. The animal was returned to its cage following treatment. At 1,24, 48, and 72 hours after dosing, the test eye of each rabbit wasexamined with an auxiliary light source for ocular irritation. Under theconditions of this study, the solution of the invention was notconsidered an irritant to the ocular tissue of the rabbit.

Skin Irritation Experiment

The solution of the invention was also evaluated for primary skinirritation based on the requirements of the International Organizationfor Standardization 10993: Biological Evaluation of Medical Devices,Part 10: Tests for Irritation and Sensitization. In cumulative skinirritation studies, two intact and two abraded skin sites were preparedon the skin on the back of each of six animals. Then, 0.5 mL of thesolution of the present invention was applied to one intact skin siteand one abraded skin site on each animal for 4 hours a day for a periodof 5 days; 0.5 mL of distilled water was applied to the second intactskin site and the second abraded skin site on each animal for the sametime period, as a control. No cumulative skin irritation effects werenoted at the application sites of the solution of the invention comparedto the distilled water.

We have studied the antimicrobial properties of the solutions of thepresent invention as well as the behavior of these solutions withrespect to eye and skin irritation and find the following results:

Skin irritation index of the solution of invention was zero as it wascompared to sterile saline.

Eye irritation index of the solution of invention was also zero as itwas compared to sterile saline.

IV. Methods for Using the Composition of the Invention

Application of the stable aqueous physiologically-balanced,non-cytotoxic ionized acidic solution of the present invention, has beendemonstrated to help wound healing progress remarkably. Antimicrobialproperties of acidic salt solutions of the invention are such that theyenhance the healing process of any wound contaminated withmicroorganisms. The compositions of the invention function specificallyto maintain the necessary antimicrobial environment for wounds to healfaster, without the usual complications associated with superficialinfections. In addition, the solutions provide topical microbial controland humidification of chronic wounds.

The use of acidic salt solutions of the present invention has beeninstrumental in healing a number of patients with deep wounds which werenot responding to usual medications and locally applied treatments. Inone aspect, the present invention provides a method for the treatment ofvarious medical conditions such as promoting wound healing, reduction ofpathogens in open wounds, wound decontamination, ocular disinfection ordecontamination, oral disinfection, antifungal therapy, ophthalmicapplications, reduction of pathogens in pulmonary infections, reductionof pathogens in burns, lavage, reduction of infectious load in organsfor transplantation, reduction of bacterial load in autologous orartificial tissue transplantation, oral disinfection antifungal therapy,treatment of biofilm for cystic fibrosis and related diseases, treatmentof viral infections, treatment of skin diseases, and tissue repair andregeneration, which method comprises using the solution of the presentinvention by applying the solution to the site where treatment isrequired. Non-limiting examples of biofilm that may be treated using thesolutions of the present invention include those cited in the reviewarticle entitled “Is there a role for quorum signals in bacterialbiofilms?” by S. Kjelleberg, and S. Molin, PMID: 12057677(PubMed-indexed for MEDLINE).

The physiologically balanced, solutions of the invention may beeffective in reducing bacterial load thus improving wound healing.Preliminary product development studies in human patients with chronicwounds suggest that solutions are well tolerated, improve thegranulation of wound tissue, reduce the need for debridement compared toprior art solutions with patients reporting less pain during theirtreatment. In addition, preliminary product development studies suggestthat, when solutions are applied to patients with artificial skin graftsthat are infected and normally need surgical replacement of the graft,the infections are eliminated and the grafts are saved.

Three recent case studies involving the treatment of human subjects witha preferred composition of the invention are presented below. In thesecase studies, the acidic salt solution was essentially the same as thatdescribed for the solution in Table 2. This composition providesosmolarity compatible with blood plasma.

The wounds were kept continuously moist with the composition of theinvention, and were covered with Vaseline gauze to prevent evaporationof the solution.

Case Study #1

The patient was a 70 year-old female, with a long history of severevenous edema, lymphaedema, and obesity. Her vascular supply was normal.She developed a cutaneous ulcer 2 years ago in the lower right leg. Asecond ulcer subsequently developed in the lateral right leg. The ulcershad previously been treated using multiple methods, includingdebridement, antibiotics, topical solutions including BETADINE® (PurdueFrederick, Norwalk, Conn.); SILVADINE® (BASF Corporation, Mt. Olive,N.J.); ELASE® (Fujisawa Co., Deerfield, Ill.); and FURACIN® (RobertsPharmaceutical Corp., Meridian Center, Illinois). By way of explanation,BETADINE® is an antiseptic cleanser, used externally on wounds; aniodine-containing preparation used as a broad spectrum antimicrobial.SILVADINE® is a soft white cream containing 1% silver sulfadiazineantimicrobial agent which is applied to wounds after cleaning anddebriding. ELASE® is an enzymatic powder or cream used as a debridementagent in wounds where circulation is poor, to destroy dead tissue andleave healthy tissue intact. FURACIN® is a nitrofurazone broad-spectrumantibacterial cream used against pathogens commonly causing surfaceinfections. Use of these agents in the wound healing had not producedthe desired results.

A biopsy revealed benign ulceration and granulation tissue. Thepossibility of Pyodermo Gangrenosum was considered. The initialmeasurements of these severely necrotic ulcers were 130×180 mm and98×125 mm. Treatment included bedrest, debridement, antibiotics, andtopical application of the composition of the invention made accordingto experimental detailed previously in Example #1, for hydration andtopical bacterial control. Within 10 days, the ulcers were almostcompletely covered with crisp red granulation tissue and the pain wasgone. Within 14 days, a split thickness skin graft closed the wound; thepatient was able to leave the hospital 8 days later. Within two monthsfollowing the start of treatment, the ulcers had completely healed, andthe patient remained pain-free.

Case Study #2

The patient was a 50 year-old male, with a history of thrombophlebitis,pulmonary emboli, and obesity. The patient had experienced infectedhematomatous ulceration in both groins and bilateral venous ulcers inboth legs for several months. He had an antithrombin III deficiency andhad been coumadinized. By way of explanation, Antithrombin III is aprotein consisting of normal plasma and extracellular sites thatinactivates thrombin in a time-dependent irreversible reaction andserves as a cofactor of heparin into its anticoagulant activities.Antithrombin III also inhibits certain coagulation factors-occurs incertain disease process i.e., liver disease or may be genetic.Coumadinized refers to the use of crystalline warfarin tabs or HeparinI.V. Anticoagulant to treat patients who have thrombosis to preventfurther thrombus. COUMADINE® is manufactured by DuPont® Pharmaceutical,Wilmington, Del. Because of the recent hemorrhages in his groin, hedeveloped large deep ulcerations on the right (measuring 140×90 mm) andmore superficial ulcerations on the left (50×50 mm and 60×60 mm). Afterthe first debridement of infected necrotic fat, the culture revealed thepresence of vancomycin-resistant Enterococcus. Treatment consisting oftopical application of the composition of the invention was started.Infectious disease consultation recommended no further antibiotictreatment. Topical dressings consisting of sponges soaked with thecomposition of the invention were packed into the wound and the patientwas subjected to bedrest. The distal venous ulcers healed fairly rapidlyand required only two more debridements. The left groin ulcer underminedand required opening further while the packing was soaked with thecomposition of the invention. The patient then began healing, with goodgranulation tissue forming and epidermal coverage to 90% in the rightgroin ulcer. The left groin ulcer required debridement for undermining,but began healing without antibiotic treatment.

Case Study #3

The patient was a 57 year-old male, who had experienced recurrent ulcersof both feet and ankles over the past four years. Local wound care hadinitially been started by coagulating veins and using topical woundtherapy. His UNNA® boots caused an increase in his ulcerations, whichthen became more severe. By way of explanation, an UNNA® boot is anelastic adhesive bandage applied over zinc oxide cream as a protectivetreatment. An UNNA® boot is a boot-like dressing of the lower extremitymade of layers of gauze and UNNA®'s paste; 100% soft cotton gauzeimpregnated with non-hardening zinc oxide paste. The manufacturer ofUNNA®'s paste is Glenwood, Inc. of Tenalty, N.J. He had been using aJOBST® pump for edema control. This pump is designed for intermittenthome use and is connected to an inflatable pneumatic appliance which istypically preset to alternate 90 seconds of inflation with 30 seconds ofdeflation. The manufacturer of JOBST® pumps is Nutech, of San Antonio,Tex.

At the time we examined the patient, his wound measurements were 33×65×2mm, 17×25×2 mm, and 5×9×2 mm. Physical evaluation verified excellentpulsatile inflow to the leg; the wounds were therefore diagnosed asvenous ulcers because of the significant edema present. The patientbegan compression therapy and debridement, culturing the leg at the sametime; the bacteria present were found to be coagulase-negative,methicillin-resistant Staphylococcus and Enterococcus sensitive tovancomycin. He also had Haemophilus and diphtheroids cultured withpolymicrobial infection. The patient had persistent nonhealinginfections for several months, and the infections had become resistantto the classic antibiotic treatments. The infections were only sensitiveto CIPROFLOXACIN® and BACTRIM DS®. CIPROFLOXACIN® is a broad spectrumantibiotic, manufactured by Miles Pharmaceutical, West Haven, Conn.,which is active on Gram+ and Gram− bacteria, and is typically used totreat skin, bone and joint infections. BACTRIM DS® is manufactured byRoche of Nutley, N.J. BACTRIM DS® is a sulfonamide antibiotic, which istypically used to treat urinary tract infections, and is also used totreat E. coli, Proteus species, Shegellosis and Pneumocystic pneumoniainfections. The patient was started on CIPROFLOXACIN®, which was thendiscontinued, and then BACTRIM DS® was started. He had topicaldebridements.

Since no significant improvement was shown after the treatment describedabove, topical application of the composition of the invention was begunfor control of the bacteria and hydration. The infections were rapidlycontrolled after the start of treatment with the composition of theinvention, and the wounds began healing fairly rapidly. He has now shownhealing of the two ulcers, with the final measurements down to 7×41 mmand 7×11 mm on the right medial and lateral ankle, respectively.

Oral Care

The physiologically-balanced, acidic solution of the invention may beused to treat canker sores (mouth ulcers) or cold sores by rinsing theaffected area. The solution can be used by soaking the cold sore 3-4times a day, each time with 2-3 applications, and putting the solutionin contact with the sore for 20-30 seconds. The solution may also beused as a mouth rinse for dental and mouth hygiene and to controlinfection. In this instance, the solution may be used as a garglingsolution to fight throat infection. The solution may be applied with thehelp of a cotton swab for more specific areas. The solution can be usedonce or several times a day according to patient's needs and condition.

Ophthalmic Care

The physiologically-balanced, acidic solution of the invention may beused in place of a saline solution to remove a foreign body from, torinse, or to irrigate the eyes. It can also be applied topically beforeor after surgery to disinfect an eye and surrounding tissues. Ourstudies on rabbits eyes showed that this solution is as safe as salinesolution when applied to rabbits' eyes and has no toxicity to the eyeswhen compared to ophthalmic grade BETADINE® (5%) typically usedpre-surgery. The solution can be used once or several times a dayaccording to a patient's needs and condition. The solution can beapplied by dropping it directly into the eyes as necessary. It can alsobe applied by soaking a gauze and applying the saturated gauze to theeyes for 1 or several minutes. It can also be used to clean the eyes bygently wiping the eyes with a saturated gauze. The solution can also bepoured into a small eye washer, then the washer is inverted over the eyewasher and the eyelid opened and closed several times.

The stable, physiologically-balanced, acidic solution of the inventionmay be used for the treatment of ocular disinfection or decontamination.In addition, it may be used as a replacement for silver nitrate in thedisinfection of the eyes of neonates. The reader will see that thesolution of the invention has applications in the treatment of manydifferent types of wounds, including, without limitation, diabeticulcers, gangrene, venous ulcers, decubitus ulcers, pressure ulcers,wounds due to bites, acute trauma wounds, surgical wounds and burns. Thecomposition of the invention is also useful as an irrigation solution,for example, during dental, periodontal, and ophthalmic procedures. Thecomposition of the invention can also be used for pre- andpost-operative cleaning of tissue sites, and as a gargling solution fortreatment of canker sores. In addition, the HOCl contained in thesolution may stimulate or enhance growth factors essential for the woundhealing process. As such, the solution may find uses in many otherapplications in which disinfection and growth factor stimulation aredesirable.

Methods of Using Solution for Skin Disinfection:

The solution of the present invention may also be used to treat skinthat are infected. In a skin of a patient showing medical signs ofinfection, the solution of the present invention may be applied directlyto the area of the skin that are infected. After at least oneapplication of the solution onto the infected skin using standardmethods of application known in the art, the disinfection properties ofthe solution maybe noted.

Reduction of Pathogens in Pulmonary Infections:

The solution of the present invention may be used for the reduction ofpathogens in pulmonary infections. For example, various viral orbacterial infections may be effectively treated with the solution of thepresent invention. Non-limited examples of infections that may beeffectively treated using the solution of the present invention includeanthrax spores present in the lungs, and the reduction of pneumoniacausing bacteria in the lungs, including strep bacteria and the like.

Method of Using the Solution of Invention for Cleaning Eves inPediatrics:

The solution of the present invention may be used for the cleaning eyesin adults and in pediatrics. For example, various viral infection,bacterial infections, or pathogenic agents may be effectively treatedwith the solution of the present invention. Non-limiting examples ofpathogenic agents that have been successfully treated with the solutionof the present invention include chlamydia trachomatis, gonorrhea aswell as other viral infections.

Method of Using the Solution of Invention in Gynecology:

The solution of the present invention may be used for the treatment ofgynecological infections, such as urinary tract infections and the like.For example, various microorganisms, yeasts (e.g., Monilia, Candidaalbicans, etc . . . . ), bacterial infections, HSV-2 or other pathogenicagents may be effectively treated with the solution of the presentinvention. Optionally, the application of the solutions of the presentinvention can be used with other medications for the treatment ofgynecological infections.

Method of Wound Care:

Patients suffering from long-lasting non-healing wounds should betreated with the physiologically-balanced, acidic solution of thepresent invention on a daily basis, typically about twice a day. Thesolution of the invention, for example, having a concentration of about180 ppm of total active chlorine and a pH of 2.5 may be used in place ofa saline solution, to control infection and to help the wound healingmechanisms. The solution of the invention may be used as follows: agauze material or gauze pad is presoaked with enough solution tosaturate it and is then squeezed to remove excess solution. This removesspecies present in the gauze which would react with and reduce theeffectiveness of the solution of the invention. The gauze is wettedafter this procedure, but not soaked. Additional solution is thenapplied to completely wet the gauze, which is then immediately appliedto the wound. In the alternative, the gauze may be applied to the woundand then additional solution applied. Typically the wound site is packedwith the solution-soaked gauze, and optionally, a Vaseline gauze can beapplied on top of the packed wound to keep it moist and free ofcontaminating germs. The wound site is then wrapped with wound dressingsas is standard in the art. The solution may also be used to clean awound by pouring it directly on the wound site to remove any necrotictissue by a mechanical procedure, and also as a cleanser or irrigant.

The patient may also make use of a “wound care kit” which permits thepatient to periodically pour the solution of the present invention ontothe wound site without having to remove the dressing. This kit providesease-of-use, portability and dramatically reduce exposure of the wound.The wound care kit includes a package containing the solution of theinvention and bandaging material.

Preferably the kit contains a package containing the solution of theinvention and a specialized bandage for use in combination with thesolution. The specialized bandage keeps the skin surrounding the wounddry while the wound is treated. Further, the bandage may be applied in aphysician's office or at a hospital, with the patient continuing care athome; may be applied and used at home under the instructions of aphysician; or for minor injuries, the wound care kit may be used as an“over the counter” treatment by the patient alone.

In another aspect of the invention, the solutions of the presentinvention may be packaged to contain the solution in individual, singleuse containers. The single-use containers may be used for example, forapplication in single change of dressing or equivalents thereof. Thesingle-use containers of the present invention may be used inconjunction with the specialized bandages disclosed in the presentinvention. In another aspect of the invention, a wound care kit maycomprise single-use containers of the solutions of the present inventionwith the specialized bandages for various applications disclosed herein.

IV. Description of the Wound Care Kit

The wound care kit includes bandaging material and a package of thesolution of the invention. Preferably the packaging material providesthe kind of non-reactive (with the solution) surface previouslydescribed herein. In addition, the bandaging material preferablyincludes a specially designed wound “bandage” made out of anoxygen-permeable bandage material to prevent the wounded tissue fromdrying. FIGS. 2A-2C and FIG. 3 describe the bandage and illustrate theuse of the bandage on a wound surface, respectively. The bandage isdescribed in more detail subsequently. The kit may also include gauze ora similar material for packing of the wound, to be used in combinationwith the solution and a bandage.

V. Description of the Specialized Bandage

The specialized bandage of the present invention comprises an opening,which may also be described as a “window” through which the solution ofthe invention or other topical material may be applied periodically asneeded depending on the indication. Preferably, the bandage includes adew/moisture sensor, an electrically-conductive sensor which measuresion content, or other bandage property sensor which provides anindication of the status of the bandage related to treatment of thewound. For example, and not by way of limitation, a dew/moistureindicator which provides a colored indication when the bandage solutioncontent has become low, or a signal-producing device such as a soundindicator or an electrical signal output indicator when the ion contentof the treatment solution has become low so that the bandage is nolonger sufficiently effective.

One embodiment of the bandage invention is shown in FIGS. 2A-2C, Thebandage 200 includes an outer portion 201 having a primary adhesiveborder 202; an inner portion 210 including a lifting flap 205 having asecondary adhesive border 207, a lifting tab 204, which assists in thelifting of flap 205, and a hinge 206. Optionally the bandage has adew/humidity indicator 208, or an electrically-conductive sensor, wherethe sensor may be attached to a signal generator, which occupies aposition within inner portion 210 of bandage 200. FIG. 2B is a schematicside view of air-permeable bandage 200, showing lifting flap 205 andlifting tab 204 in a partially lifted position, to provide a windowopening 203 through bandage 200. A portion of secondary adhesive border207 has been lifted above the upper surface 209 of bandage 200. FIG. 2Cis a schematic cross-sectional view of air-permeable bandage 200, withlifting flap 205 and lifting tab 204 in a lowered position, secured toupper surface 209 of bandage 200 by secondary adhesive border 207. Oneskilled in the art can envision a number of similar designs which willaccomplish the function and utility of the bandage in a similar manner,and such designs are considered to be included in the present invention.

FIG. 3 is a schematic cross-sectional view 300 of an air-permeablebandage 200 of the kind shown in FIGS. 2A-2C, applied over asubcutaneous wound 303. The subcutaneous tissue 304 is packed with apacking material 306 such as gauze, which has been treated to reduce oreliminate reactivity with oxidants and then soaked in thephysiologically balanced, electrolyzed, acidic solution 308 of thepresent invention. The bandage 200 is adhered to the skin surface 302 bya primary adhesive border 202. Bandage lifting flap 205 can be liftedvia tab 204 to expose packing material 306 for the application ofadditional solution 308 when desired. A dew/humidity indicator (notshown), or electrically-conductive indicator (not shown) may be used toindicate the appropriate time for addition of solution 308.

In another aspect of the bandage invention, the window may have nopermanent connecting hinge with the bandage and may be removablyattached or secured to the bandage by various connecting or attachingmeans known in the art. Non-limiting examples of such connecting orattaching means include Velcro attachments, removable adhesives ortacking surfaces. The removable windows in the bandages of the presentinvention permit the changing or replacement of the windows without theneed for replacing the entire outer bandage.

In another aspect of the bandage invention, individual bandages ofvariable sizes and configurations may be supplied and sold separatelywith the corresponding detachable windows of particular sizes andconfigurations that may accommodate the bandage. Optionally, the windowsmay be designed such that the windows properly overlaps with the bandagesuch that the windows fully cover the wounds and have overlappingsurfaces with the bandage such that the windows may be securely attachedto the bandage.

The bandage provides ease-of-use to the patient by allowing the patientto pour the solution onto his wound or onto wound packing without havingto remove the entire dressing. A more complicated version of thebandage, such as one having an electrically-conductive sensor which maybe connected to monitoring equipment is particularly helpful in ahospital setting.

In another aspect of the bandage invention, the bandages of variablesizes, contours and shapes may be pre-fabricated with perforatedoutlines of one or more windows of variable sizes, dimensions andconfigurations such that the bandage may be adapted or custom fitted tothe size, shape and configuration of the wounds. The bandages maybedesigned for variable sizes, shapes, and contours that can be made toaccommodate the specific anatomical dimensions of the body. Particularareas of the body that may require specially designed bandages includevarious joints, the elbows, knees, fingers and toes, and other locationsof the human anatomy having non-flat surfaces or curves.

The perforations in the bandages allows a health caregiver or thepatient to use a generically manufactured, perforated bandage tomanually remove the inner window of the bandage by cutting or tearingalong the perforations defining or outlining a window that custommatches the size, shape or configuration of the wound. In addition, theperforations permit the application of a single bandage for applicationto wounds of various sizes and dimensions, and permit medical facilitiesand suppliers to stock only a small number of intermediate sizes ofbandages that may accommodate wounds of various sizes andconfigurations.

ASPECTS OF THE INVENTION

In one aspect of the invention, there is provided a stable aqueousphysiologically balanced, ionized solution comprising: (a) an acidicsolution of hypohalous acid with a concentration from about 10 ppm toabout 200 ppm; (b) a halide comprising salt, from about 0.4 g/L to about20.4 g/L, said solution having a pH range from about 3.0 to about 4.0,and (c) said solution when stored in a chemically non-reactive containerat room temperature over at least three months, the solution ischaracterized as having a reduced hypohalous acid concentration ofbetween 1-95% as determined by UV-VIS or by iodometric titration.

In another aspect of the invention, the solution is furthercharacterized as having a pH range from about 3.5 to about 4.0. Inanother aspect, the stable solution has a pH of about 3.5.

In another aspect, the solution is further characterized as capable ofbeing stored in a chemically non-reactive container at room temperatureover at least three months, the solution is characterized as having areduced hypohalous acid concentration of between 0-5% as determined byUV-VIS or by iodometric titration.

In yet another aspect, the stable solution may be stored in a chemicallynon-reactive container at room temperature over at least three months,the solution is characterized as having a reduced hypohalous acidconcentration of between 5-15% as determined by UV-VIS or by iodometrictitration.

In another aspect of the invention, the hypohalous acid comprises theactive chlorine species HOCl, NaOCl, and Cl₂, and the pH of the solutionis about 3.5.

In one aspect of the invention, there is provided a stable aqueousphysiologically balanced, ionized solution, wherein the chemicallynon-reactive container is a gas-tight, sealed container is made frommaterial that is non-permeable and resistant to oxidative degradation.

In another aspect, the chemically non-reactive container is a gas-tight,sealed container made from PFA or equivalent PFA compositions or Tefloncompositions. In another aspect, the container is non-permeable to thespecies of the NVC-101 solution.

In yet another aspect, the stable solution is further characterized ashaving an original oxidation reduction potential (ORP) at roomtemperature ranging from about +600 mV to about +1200 mV, and said ORPranging from no less than about 90 to 97.5% of the original ORP afterthree months at room temperature.

In one aspect, the hypohalous acid is selected from the group consistingof HOBr, HOI, HOCl, and HOF. In another aspect, the hypohalous acidconcentration is from about 40 to about 190 ppm.

In yet another aspect, the halide comprising salt is a member selectedfrom the group consisting of lithium, sodium, potassium, magnesium,zinc, cesium, rubidium, and barium halide.

In yet another aspect, the halide comprising salt is a single salt. Inyet another aspect, the hypohalous acid concentration of the stablesolution is measured by iodometric titration or UV-VIS spectroscopy.

In another aspect, the chemically non-reactive container is designed forsingle use or single application packaging.

In a further aspect, there is provided a stable aqueous physiologicallybalanced, ionized solution comprising: (a) an acidic solution ofhypochlorous acid (HOCl) with a concentration, from about 10 ppm toabout 200 ppm; (b) a chloride comprising salt, from about 0.4 g/L toabout 16 g/L, said solution having a pH range from about 3.5 to about4.0, and (c) said solution when being stored in a chemicallynon-reactive, single use container at room temperature over at leastthree months and having a reduced hypochlorous acid concentration ofbetween 1-95% as determined by UV-VIS or by iodometric titration.

In one aspect, the container is a single use PFA lined container.

In one aspect, there is provided a method for the treatment of variousmedical conditions selected from the groups consisting of promotingwound healing, reduction of pathogens in open wounds, wounddecontamination, ocular disinfection or decontamination, oraldisinfection, antifungal therapy, ophthalmic applications, reduction ofpathogens in pulmonary infections, reduction of pathogens in burns,lavage, reduction of infectious load in organs for transplantation,reduction of bacterial load in autologous or artificial tissuetransplantation, oral disinfection antifungal therapy, treatment ofbiofilm for cystic fibrosis or other diseases that produces biofilms,treatment of viral infections, treatment of skin diseases, and tissuerepair and regeneration, which method comprises using a solution of theinvention by applying the solution to the site where treatment isrequired.

In yet another aspect, there is provided a method for the treatment ofvarious medical conditions selected from the groups consisting ofpromoting wound healing, reduction of pathogens in open wounds, wounddecontamination, ocular disinfection or decontamination, oraldisinfection, antifungal therapy, ophthalmic applications, reduction ofpathogens in pulmonary infections, reduction of pathogens in burns,lavage, reduction of infectious load in organs for transplantation,reduction of bacterial load in autologous or artificial tissuetransplantation, oral disinfection antifungal therapy, treatment ofbiofilm for cystic fibrosis or other diseases that produces biofilms,treatment of viral infections, treatment of skin diseases, and tissuerepair and regeneration, which method comprises using a solution of theinvention by applying the solution to the site where treatment isrequired.

In another aspect, the solution is characterized as having an originaloxidation reduction potential (ORP) at room temperature ranging fromabout +600 mV to about +1200 mV, and said ORP ranging from no less thanabout 90 to 97.5% of the original ORP after at least three months atroom temperature.

In a further aspect, there is provided a solution wherein thehypochlorous acid concentration is from about 40 to about 190 ppm. Inanother aspect, the chloride comprising salt is a member selected fromthe group consisting of lithium, sodium, potassium, magnesium, zinc,cesium, rubidium, and barium chloride. In yet another aspect, thechloride comprising salt is a single salt.

In another aspect, there is provided a solution wherein the pH rangesfrom about 3.5 to about 4.0. In another variation of the solution, thechloride comprising salt is sodium chloride. In one aspect, theconcentration of sodium chloride is from about 4 g/L to about 9 g/L.

In one aspect of the invention, the molar ratio range of OCl⁻ over thesum of OCl⁻ and HOCl at 20° C. is about 0 to about 0.26%.

In another aspect, there is provided a method for the treatment ofvarious medical conditions selected from the groups consisting ofpromoting wound healing, reduction of pathogens in open wounds, wounddecontamination, ocular disinfection or decontamination, oraldisinfection, antifungal therapy, ophthalmic applications, reduction ofpathogens in pulmonary infections, reduction of pathogens in burns,lavage, reduction of infectious load in organs for transplantation,reduction of bacterial load in autologous or artificial tissuetransplantation, oral disinfection antifungal therapy, treatment ofbiofilm for cystic fibrosis or other diseases that produces biofilms,treatment of viral infections, treatment of skin diseases, and tissuerepair and regeneration, which method comprises using a solution of theinvention by applying the solution to the site where treatment isrequired.

In one aspect, there is provided a process for the preparation of asolution of the invention, wherein the solution is prepared by chemicalmethods, including chemical synthesis, mechanical methods such as bymixing, electrolysis or prepared in situ.

In another aspect, the halide comprising salt solution is converted toan acidic solution by electrolysis.

In yet another aspect for the process for preparing a solution of theinvention, the pH of the solution is adjusted to about 3.5 to about 4.0.In yet another aspect, the solution is prepared by chemical synthesiscomprising of the following reactions:

NaOCl+HCl→HOCl+NaCl

wherein the concentration of NaOCl in solution is about 2.5 mmol/L andthe concentration of HCl is about 7.4 mmol/L, the resulting solution isadjusted to a pH of about 3.5 to about 4.0, and the solution is allowedto reach equilibrium without agitation for at least 2 hours.

In another aspect, there is provided a process wherein the solution isprepared in situ by mixing chemicals to form the stable aqueousphysiologically balanced, non-cytotoxic ionized solution at the site ofthe tissue in need of treatment.

In yet another aspect, there is provided a method of promoting woundhealing, reduction of pathogens in open wounds, wound decontamination,ocular disinfection or decontamination, oral disinfection, antifungaltherapy, ophthalmic applications, reduction of pathogens in pulmonaryinfections, reduction of pathogens in burns, lavage, reduction ofinfectious load in organs for transplantation, reduction of bacterialload in autologous or artificial tissue transplantation, oraldisinfection antifungal therapy, treatment of biofilm for cysticfibrosis or other diseases that produces biofilms, treatment of viralinfections, treatment of skin diseases, and tissue repair andregeneration, or a combination thereof, by treating a patient in need ofsuch therapy with an effective amount of a stable,physiologically-balanced, acidic composition comprising an aqueousstable solution of the present invention. In a further aspect, there isprovided the above method that comprises a) exposing area of damagedtissue; b) applying the solution to dermal tissue; c) irrigation ofdamaged tissue using the solution; and d) cleaning or treating tissueusing the solution.

Accordingly, the above described preferred embodiments are not intendedto limit the scope of the present invention, as one skilled in the artcan, in view of the present disclosure, expand such embodiments tocorrespond with the subject matter of the invention claimed below.

1-36. (canceled)
 37. A method for the treatment of a medical conditionselected from the groups consisting of disinfection or decontaminationof open wounds and burns, promotion of wound healing, in a patient inneed of such treatment comprising: applying to a treatment site aneffective amount of a stable aqueous physiologically balanced, ionizedsolution comprising an acidic solution of hypohalous acid with aconcentration from about 0.1 ppm to about 1,000 ppm; a halide comprisingsalt, the halide comprising salt is sodium chloride at a concentrationof about four-tenth to slightly higher than full strength of normal orisotonic saline solution, said solution having a pH range from about 3.0to about 4.0, an original reduction potential (ORP) at room temperatureranging from about +600 mV to about +1200 mV, and said ORP ranging fromno less than about 90 to 97.5% of the original ORP after three months atroom temperature; and said solution when stored in a chemicallynon-reactive container at room temperature over at least three months,the solution is characterized as having a reduced hypohalous acidconcentration of between 1-95% as determined by UV-VIS or by iodometrictitration.
 38. A method for the treatment of a medical conditionselected from the groups consisting of disinfection, decontamination ofopen wounds and promotion of wound healing in a patient in need of suchtreatment comprising: applying to a treatment site an effective amountof a stable aqueous physiologically balanced, ionized solutioncomprising an acidic solution of hypohalous acid with a concentrationfrom about 1 ppm to about 750 ppm; a halide comprising salt, the halidecomprising salt is sodium chloride at a concentration of aboutfour-tenth to slightly higher than full strength of normal or isotonicsaline solution, said solution having a pH range from about 3.0 to about4.0, an original reduction potential (ORP) at room temperature rangingfrom about +600 mV to about +1200 mV, and said ORP ranging from no lessthan about 90 to 97.5% of the original ORP after three months at roomtemperature; and said solution when stored in a chemically non-reactivecontainer at room temperature over at least three months, the solutionis characterized as having a reduced hypohalous acid concentration ofbetween 1-95% as determined by UV-VIS or by iodometric titration.
 39. Amethod of disinfecting or decontaminating open wounds and promotingwound healing by treating a patient in need thereof with an effectiveamount of a stable aqueous physiologically balanced, ionized solutioncomprising an acidic solution of hypohalous acid with a concentrationfrom about 5 ppm to about 500 ppm; a halide comprising salt, the halidecomprising salt is sodium chloride at a concentration of aboutfour-tenth to slightly higher than full strength of normal or isotonicsaline solution, said solution having a pH range from about 3.0 to about4.0, an original reduction potential (ORP) at room temperature rangingfrom about +600 mV to about +1200 mV, and said ORP ranging from no lessthan about 90 to 97.5% of the original ORP after three months at roomtemperature; and said solution when stored in a chemically non-reactivecontainer at room temperature over at least three months, the solutionis characterized as having a reduced hypohalous acid concentration ofbetween 1-95% as determined by UV-VIS or by iodometric titration. 40.The method of claim 37 wherein the pH range is from about 3.5 to about4.0.
 41. The method of claim 38 wherein the pH range is from about 3.5to about 4.0.
 42. The method of claim 39 wherein the pH range is fromabout 3.5 to about 4.0.
 43. The method of claim 40 wherein the pH isabout 3.5.
 44. The method of claim 41 wherein the pH is about 3.5. 45.The method of claim 42 wherein the pH is about 3.5.
 46. The method ofclaim 37 wherein the sodium chloride concentration is from about 4 g/lto about 10 μl.
 47. The method of claim 38 wherein the sodium chlorideconcentration is from about 4 g/l to about 10 g/l.
 48. The method ofclaim 39 wherein the sodium chloride concentration is from about 4 g/lto about 10 g/l.
 49. The method of claim 46 wherein the sodium chlorideconcentration is about 9 g/l.
 50. The method of claim 47 wherein thesodium chloride concentration is about 9 g/l.
 51. The method of claim 48wherein the sodium chloride concentration is about 9 g/l.
 52. The methodof claim 37, the hypohalous acid is selected from the group consistingof HOBr and HOCl.
 53. The method of claim 38, the hypohalous acid isselected from the group consisting of HOBr and HOCl.
 54. The method ofclaim 39, the hypohalous acid is selected from the group consisting ofHOBr and HOCl.